Chronic Inhibition of Dipeptidyl Peptidase-4 with a Sitagliptin Analog Preserves Pancreatic Islet Cell Mass and Function in a Rodent

Model of Type 2 Diabetes

James Mu1, John Woods2, Yun-Ping Zhou1, Zhihua Li1, Emanuel Zycband2, Yue Feng1, Lan Zhu1 Andrew Howard1, Ranabir Sinha Roy1, Cai Li1, Nancy A. Thornberry1, Bei B. Zhang1 Metabolic Disorders-Diabetes1, Target Validation2 Merck Research Laboratories, Rahway, NJ 07065

Homeostasis Defects in Type 2 Diabetes

meal Insulin Type 2 DM Normals Delayed / depressed insulin release

-60

60

120

180

240

Glucagon

Non-suppressed glucagon

Glucose

Hyperglycemia

Muller et al NEJM 283:109, 1970

GLP-1: Regulation and Action

Meal bolus

GI Tract
80

Meal GLP-1

GLP-1 Neuroendocrine Cells in Ileum Neural innervation

Skeletal muscle

40 0

Delayed Gastric Emptying

Glucose uptake

500 400

Insulin

GLP-1
Insulin

300 200 100 0

Pancreatic islet

30

60

120

180

240

CNS

Glucagon Food Intake/BW Decreased HGP

DPP-4 regulates GLP-1 activity in vivo

7 HAagonist, active
intact GLP-1

36

total GLP-1

DPP-4
9

t1/2 ~ 1 min
36

inactive
Holst et al. Diabetes 47, 1663-1670 (1998)

DPP-4 inhibitors provide an oral approach to GLP-1-based therapy

Sitagliptin: A Potent and Selective DPP-4 Inhibitor

F F NH2 O N F N N N CF3

enzyme Dipeptidyl peptidase 4 Dipeptidyl peptidase 8 Dipeptidyl peptidase 9 Quiescent cell prolyl peptidase (DPP7, DPP II) Fibroblast activation protein (seprase) Prolyl endopeptidase Aminopeptidase P

IC50 (nM) 18 48,000 >100,000 >100,000 >100,000 >100,000 >100,000

Preparation of ICR HFD/STZ Mouse Model

ICR mice were put on HFD at 4 week of age and were left on HFD diet for 3 weeks before they were injected with streptozotocin at 100mg/kg. Mice were used for study after 3 more weeks when blood glucose reached ~300mg/dL. Data shown in table are mean SEM, n = 8 -10. * p < 0.05 comparing control and treated group.

Week 4
Chow HFD

7
HFD/STZ

10

Testing
HFD + compounds

Blood glucose (mg/dl)

350 300 250 200 150 100 50 0
al on e al on e nt ro l /S TZ FD co Z FD

Plasma insulin (ng/ml)

*

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

al on e nt ro l on e al ST Z FD FD H co /S TZ

ST

Le an

Le an

Des-fluoro-sitagliptin Treatment Improved Glycemic Control

Ambient Glucose (mg/dl)

6 hour Fasting Glucose (mg/dl)

A. 400
300 200 100 0

B.

400

* * * * * * * * * * * * * * * * * * * *

*
300 200 100 0 2.0

* * *

10 11 12

6.0

11.0

Weeks of treatment Diabetic control Des-F-sitagliptin 0.1% Des-F-sitagliptin 0.4% Des-F-sitagliptin 1.1% Non-diabetic control
* p<0.05 vs. diabetic control

Weeks of treatment

HbA1c (%)
C.
7 6 5 4 3 2 1 0 0 8

* *

Weeks of treatment

Effect of Des-fluoro-sitagliptin on Dyslipidemia

B. 15
10

A.
4 3

Liver Weight (g)

Liver TG (mg/g)

*
2

* *

5
1 0

800

C.
600 400 200 0

Plasma TG (mg/dl) * * *

D. 6
5 4 3 2 1 0

Plasma FFA (mM)

Diabetic control Des-F-sitagliptin (0.1%) Des-F-sitagliptin (0.4%)

Des-F-sitagliptin (1.1%) Non-diabetic control

* p<0.05 vs. diabetic control

Effect of Des-F-sitagliptin on Islet Cell Mass

A
Nondiabetic Control H&E insulin (I) glucagon (G) I/G

B.
0.6

-Cell / Total Islet Area

0.4

Diabetic Control

0.2

0.0

Diabetic Mice Treated with Des-F-sitagliptin 0.1%

C.

0.3

Cell / Total Islet Area

0.2

*
0.4%
0.1

*
1% 4%

*
1%

*
ro l co nt

0.0

co nt ro l

be t

N on
* p<0.05 vs. diabetic control

-d ia

D ia

be tic

1.1%

Des-F-sitagliptin

ic

1.

0.

0.

Comparison of Des-F-sitagliptin, Rosiglitazone and Glipizide on Glycemic Control

A.
400 300 200 100 0 -1.0

Ambient Glucose (mg/dl) * * * * * * * * * * ** * * ** * * * * ** ** ** **

Dabetic control

B.

55 50 45 40 35

Body Weight (g) * * * * *

* * *

Des-F-sitagliptin 1.1% Rosiglitozone 0.01% Glipizide 0.02% Non-diabetic control

1.5

4.0

6.5

9.0

30 -1.0

1.5

4.0

6.5

9.0

Time (week)

C.
350 300 250 200 150 100 50 0

Time (week)

6 hour Fasting Glucose (mg/dl) * * *

* * *

D.
700 600 500 400 300 200 100 0

OGTT Glucose (mg/dl)

* *

* *

* * * * * * 0 * * * 25 50 75 100 *

9
Rosiglitazone 0.01% Glipizide 0.02%

Time (Week)
Diabetic control Des-F-sitagliptin 1.1% Non-diabetic control

Time (Min)
* p<0.05 vs. diabetic control

Effects of Des-F-sitagliptin, Rosiglitazone and Glipizide on Plasma Parameters

A.
HbA1c (%)
8 7 6 5 4 3 2 1 0

B.
* * * *

Intact GLP-1 (pM)

14 12 10 8 6 4 2 0

C.
2.0 1.5

Plsama Insulin (ng/ml)

D.120
100 80 60 40

Plasma Glucagon (pg/ml)

*
1.0 0.5

20

0.0

Diabetic control Des-F-sitagliptin 1.1% Rosiglitazone 0.01%

Glipizide 0.02% Non-diabetic control

* p<0.05 vs. diabetic control

Effect of Des-F-sitagliptin, Rosiglitazone and Glipizide on Tissue Mass and Liver Triglyceride
A. 12
11 10 9 8 7 6 5 4 3 2 1 0

Fat mass (g) *

B. 35
30 25 20 15

Muscle mass (g)

* *

10 5 0

C.
7 6 5 4 3 2 1 0

Liver TG (mg/g liver)

D.3.5
3.0 2.5 2.0

Liver weight (g)

* * * *

1.5 1.0 0.5 0.0

Diabetic control Des-F-sitagliptin 1.1% Rosiglitazone 0.01%

Glipizide 0.02% Non-diabetic control

* p<0.05 vs. diabetic control

Effects of Chronic Des-F-sitagliptin, Rosiglitazone and Glipizide Treatment on Islet Morphology

A.

Diabetic control

Nondiabetic control

Des-F-sitaglipitin

Rosiglitazone

Glipizide

B.

cell / total islet area

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
Diabetic control

* *
**

C.
0.5 0.4 0.3 0.2 0.1

cell / total islet area

* *
Des-F-sitagliptin Glipizide

Des-F-sitagliptin

Glipizide

0.0
Diabetic control

Nondiabetic control

Rosiglitazone

Nondiabetic control

Rosiglitazone

* p<0.003 vs. diabetic control. ** p=0.01

Effects of Chronic Des-F-sitagliptin, Rosiglitazone and Glipizide Treatment on Islet Function

A.
Islet insulin (ng/islet)
175 150 125 100 75 50 25 0 * * * *

B.

17.5 15.0 12.5 10.0 7.5 5.0 2.5 0.0

KCl Stimulated Islet insulin Secretion (ng/ml)

* * *

C.
Insulin Secretion (ng/ml)

12.5

Glucose Stimulated Insulin Secretion

*

10.0

*
Diabetic control

7.5 * * 2.5 * 0.0 2 * *

Non-diabetic control Des-F-sitagliptin Rosiglitazone Glipizide

* p<0.05 vs. diabetic control

5.0

Glucose (mM)

16

Summary
Chronic treatment with the des-fluoro-sitagliptin in a mouse model of type 2 diabetes with defects in insulin sensitivity and secretion resulted in significant and dose-dependent correction of postprandial and 6-hour fasting hyperglycemia. Reduction of HbA1c, circulating triglyceride, free fatty acid, liver triglyceride and hepatomegaly was also observed. The beneficial effect of the DPP-4 inhibitor on glycemic control is associated with significantly increased -cell mass and function. This is also accompanied by lower -cell mass and glucagon level. DPP-4 inhibition with des-fluoro-sitagliptin demonstrated superior glucose lowering efficacy and islet cell preserving effects compared to glipizide. Unlike Rosiglitazone treatment, chronic DPP-4 inhibition is not associated with body weight gain. This study suggests the beneficial effects of DPP-4 inhibition could extend beyond glycemic regulation via modulation of insulin secretion to include a potential reduction in cell failure commonly observed in diabetic patients.